WO2021003952A1

WO2021003952A1 - Method for sequencing rna fragments protected by trace amount of cell ribosome

Info

Publication number: WO2021003952A1
Application number: PCT/CN2019/120536
Authority: WO
Inventors: 頡伟; 熊竹清
Original assignee: 清华大学
Priority date: 2019-07-08
Filing date: 2019-11-25
Publication date: 2021-01-14
Also published as: CN112195226B; CN112195226A

Abstract

Proposed in the present invention is a method for sequencing RNA fragments protected by a trace amount of cell ribosome. The method comprises (1) lysing cells to obtain a lysate; (2) performing an enzymolysis on the lysate by using ribonuclease and adding the obtained enzymolysis solution to a surface of a sucrose buffer, centrifuging, and collecting a precipitate; (3) purifying the precipitate to obtain a ribosome-protected RNA crude extract; (4) performing electrophoresis on the ribosome-protected RNA crude extract, collecting gel slices in a predetermined area, and extracting ribosome-protected RNA from the gel slices; and (5) constructing a library and sequencing the ribosome-protected RNA. In step (1), the number of cells is 50 to 1×10⁶.

Description

Method for sequencing RNA fragments protected by very small amounts of ribosomes

Technical field

The invention relates to the field of biology. Specifically, the present invention relates to a method for sequencing a very small amount of RNA fragments protected by ribosomes.

Background technique

In recent years, with the development of second-generation sequencing technology, more technologies have emerged to help people understand various events in biology. For gene expression regulation, a large number of omics technologies based on measuring mRNA levels have emerged, while related omics methods for measuring other key steps in gene expression regulation are relatively rare. Translation is an important part of gene expression. In this process, ribosomes translate mRNA into polypeptides, which are further folded and modified into active proteins. In order to be able to produce the right protein in the right place for the cell, the cell consumes a huge amount of energy to regulate this process. Misregulation of translation will bring diseases to humans, such as neurodegenerative diseases and anemia. Therefore, understanding the translation groups in different cells and different developmental processes will help understand biological phenomena and help the treatment of diseases.

At present, the research and development of ribosome mapping technology has realized the measurement of the translation level of the whole genome. The core of the ribosome mapping technology is that a ribosome being translated occupies a -30 nt section of the mRNA. This occupation is so strong that it can protect them from being digested by nucleases. These ribosome-protected fragments are sequenced to determine the precise location of the ribosome being translated. Subsequently, people discovered many new or selectively translated protein products. However, due to technical limitations, the number of cells currently requires more than ¹⁰⁷ to be tested. For some rare cell types, this number of cells cannot be collected. The traditional ribosome mapping technology requires many times of electrophoresis gel purification due to the cumbersome steps, and the construction of small RNA libraries has always been a difficult point in the field, resulting in a situation where a small number of cells cannot be tested.

Therefore, the method for sequencing RNA fragments protected by ribosomes remains to be studied.

Summary of the invention

The present invention aims to solve at least one of the technical problems existing in the prior art to a certain extent. For this reason, the present invention proposes a method for sequencing RNA fragments protected by ribosomes, which can realize RNA fragments protected by ribosomes using a very small number of cells (as low as dozens) in a relatively short time. Sequencing to obtain high-quality genome-wide translation group data provides the possibility to explain the regulation of a small number of cellular translation levels.

In one aspect of the present invention, the present invention provides a method for sequencing RNA fragments protected by ribosomes. According to an embodiment of the present invention, the method includes: (1) lysing the cells to obtain a lysate; (2) using ribonuclease to digest the lysate, and digest the obtained enzyme The solution is added to the surface of the sucrose buffer, centrifuged to collect the precipitate; (3) the precipitate is purified to obtain a ribosome-protected RNA crude extract; (4) the crude extract is subjected to electrophoresis , Collecting a gel slice in a predetermined area, and extracting ribosome-protected RNA from the gel slice; and (5) constructing and sequencing the ribosome-protected RNA; wherein, in step (1), The number of the cells is 50 to 1×10 ⁶ cells.

Using the traditional method of building a database needs to be tested to more than ¹⁰⁷ the number of cells, however, for some rare cell types, such a large number of cells are difficult to collect. Furthermore, the inventors optimized the extraction process of ribosome-protected RNA fragments. By using the above steps, a very small number of cells can be used to sequence the ribosome-protected RNA fragments and obtain high-quality genome-wide translations. Group data, which provides the possibility to explain the regulation of a small number of cellular translation levels. In addition, the method has simple steps and can be completed in a short time (such as 3 days), reduces the cost of the experiment, improves the efficiency of the experiment, and has broad application prospects.

According to an embodiment of the present invention, the above-mentioned ribosome-protected RNA fragment sequencing method may also have the following additional technical features:

According to an embodiment of the present invention, in step (2), RNase I enzyme is used to digest the lysate, wherein the amount of RNase I enzyme is 0.5-1.5 μL, preferably 1.0 μL. As a result, RNase I enzyme is used to degrade the RNA in the lysate, while the RNA fragments protected by the ribosomes are retained.

In the enzyme digestion reaction, the addition of excessive enzymes will often lead to over-digestion, which leads to the inability to obtain enough fragments for the next experiment, while too little enzyme amount makes the reaction incomplete, and the obtained fragments are not exactly the desired purpose. Fragment, leading to affect the experimental results. The inventors conducted experiments with 10,000 mouse embryonic stem cells and 1,000 mouse embryonic stem cells, adding 2μL, 1μL, 0.5μL, and 0.1μL, respectively. The experiment results showed that the results of 0.5μL and 1μL It is obviously better than 2μL or 0.1μL, and the results are more consistent in the two cell numbers.

According to an embodiment of the present invention, step (2) further includes: adding the sucrose buffer solution to a centrifuge tube, and then transferring the enzyme digestion solution to the surface of the sucrose buffer solution at a temperature of 200,000～2～6℃. Centrifuge at a rotation speed of 350,000g for 2-6 hours, aspirate the supernatant from the centrifuge tube, wash the precipitate with agglomerate buffer until the precipitate is dissolved; wherein the agglomerate buffer includes: a concentration of 0.5 to 1.5% by mass of SDS It is a Tris buffer with a pH of 7-8 at 5-15mM. Ribosomes can be precipitated in the above-mentioned sucrose buffer, thereby achieving the purpose of separation. However, the precipitated ribosomes will stick to the side wall of the centrifuge tube after centrifugation, which is not convenient for subsequent transfer. Furthermore, the inventors have found through a lot of experiments that the adherent ribosome precipitate can be separated from the tube wall under the soaking solution containing the above-mentioned clump buffer, thereby facilitating subsequent operations.

According to an embodiment of the present invention, in step (3), the purification treatment includes: mixing and standing the precipitate with TRIzol and chloroform in sequence, and subjecting the resulting mixture to centrifugation to collect the supernatant ; Add isopropanol and glycogen to the supernatant, let stand, centrifuge, discard the supernatant, suspend the precipitate with ethanol, centrifuge, discard the supernatant, dry the precipitate, and then dissolve the Precipitation to obtain a crude extract of RNA protected by the ribosome. Thus, in order to achieve the purpose of preliminary purification.

According to an embodiment of the present invention, in step (4), a gel is used for electrophoresis, each gel is loaded with a crude extract, and no marker solution is loaded. Because the amount of RNA in the crude extract is very small, in order to prevent mutual contamination of samples during the gel running process, one gel is prepared for each sample. In addition, because the concentration of RNA in the marker solution is very high, in order to avoid contamination of samples containing a small amount of RNA during the gel running process, the marker solution is not loaded. Specifically, the pre-experiment can be carried out under the same electrophoresis conditions, the marker solution can be separately loaded to determine the position of RNA of different lengths, and then the pre-experimental film can be compared with the film after the sample is run to determine the sample Film position where RNA of predetermined length is located.

According to the embodiment of the present invention, in step (5), the CAT Small RNA-seq kit of diagenode company is used to construct the database. The inventors conducted research on some published library building kits and found that the kits of this manufacturer and model can accurately and specifically realize the library building of a very small amount of RNA fragments protected by ribosomes.

In yet another aspect of the present invention, the present invention provides another method for sequencing RNA fragments protected by ribosomes. According to an embodiment of the present invention, the method includes:

(1) Provide the following reagents:

Polyribosome buffer solution, including: 20mM Tris-HCl solution, pH=7.4; 150mM NaCl solution; 5mM MgCl ₂ solution, 1mM dithiothreitol solution; 100μg/ml cycloheximide (CHX) Solution

The lysis buffer includes: 1× the polysome buffer, 1% by mass Triton-X 100 solution; 25 U/ml of deoxyribonuclease (Turbo DNase);

Sucrose buffer solution, including: 1×polyribosome buffer; 1M sucrose solution; 20U/ml RNase inhibitor (SUPERase.In);

RNA gel extraction buffer, including: 300mM sodium acetate solution; 1M NaCl solution; 0.05% by mass Tween; 0.5mM EDTA;

Agglomerate buffer, including: 1% by mass SDS solution; 10mM Tirs solution, pH=7.5;

(2) Put 50～1×10 ⁶ cells in a centrifuge tube;

(3) Add 310 μl of ice-cold lysis buffer to the centrifuge tube, mix well, invert the centrifuge tube back and forth, and then incubate the centrifuge tube on ice;

(4) Place the centrifuge tube in a centrifuge at 4°C, centrifuge at 20,000g for 10 minutes, recover the supernatant and place it in a new centrifuge tube;

(5) Add 1 μl of RNase I to the supernatant, and shake it with a constant temperature mixer at 1000 rpm for 45 minutes at 22°C;

(6) Add 10μl of RNase inhibitor, stop nuclease digestion, and obtain lysate;

(7) First take 0.7ml sucrose buffer solution and place it at the bottom of the centrifuge tube, slowly transfer 300μL of the lysate from the previous step to the top of the sucrose solution, and place the centrifuge tube in the rotor of the centrifuge at 4℃, 260,000g. Centrifuge for 4 hours to precipitate ribosomes;

(8) Remove the centrifuge tube from the rotor, discard the supernatant in the tube, wash the place where the ribosome clumps are with 50μL clump buffer until they are completely dissolved, then transfer the lysate to a new centrifuge Tube in

(9) Add 1ml of TRIzol, mix well, leave at room temperature for 5 minutes, then add 0.2ml of chloroform, after mixing, leave at room temperature for 2 minutes, centrifuge at 4℃, 12,000g for 10 minutes, collect the supernatant in a new In the centrifuge tube, add 0.75ml of isopropanol and 1μL of liver glycogen to the centrifuge tube;

(10) Place at -20°C for 30 minutes or overnight; centrifuge for 40 minutes at 4°C and maximum speed in a centrifuge, discard the liquid, float the precipitate with 80% ethanol, centrifuge for 5 minutes and discard it The supernatant, open the lid, and dry the precipitate;

(11) Dissolve the precipitate with 6μL of RNase-free water to obtain the ribosome-protected RNA crude extract;

(12) Prepare 15% urea-TBE-PAGE gel, rinse the sample hole of TBE gel, blow off excess urea, and perform electrophoresis in 1×TBE solution at 210V for 20 minutes in advance;

(13) Add 6 μL 2× denaturation loading buffer to the crude extract, and denature the sample at 80°C for 90 seconds;

(14) Carefully rinse the sample hole again, load the sample after the previous step of denaturation treatment, and only load one piece of glue for each sample, and not load the marker solution;

(15) Electrophoresis is separated under 210V voltage for 60 minutes, and electrophoresis is stopped when the dark blue dye runs out of the gel;

(16) Determine the lane where the sample is located, start from the light blue dye, cut a piece of glue, and put it into a 1.5ml centrifuge tube without RNase;

(17) Cut off the 29-34 nt RNA aggregation area on the gel block and put it into a new centrifuge tube;

(18) Add 400μL RNA gel extraction buffer to the centrifuge tube, and place it at -80 for 30 minutes;

(19) Place the centrifuge tube in a constant temperature mixer at 22°C at 1000 rpm and gently mix overnight;

(20) Centrifuge, collect the liquid at the bottom of the tube, and transfer 400 μL of liquid to a clean centrifuge tube;

(21) Add 500 μL of isopropanol to the centrifuge tube, then add 1 μL of glycogen, and mix upside down, and repeat steps (10) and (11) to purify RNA;

(22) Use diagenode's CAT Small RNA-seq kit to build a library of dissolved RNA and sequence the obtained library.

In the method of the present invention, after the improvement and simplification of various steps, the traditional 7 days can be shortened to 3 days, and a very small number of cells (as low as dozens) are used to obtain high-quality genome-wide The translation group data provides the possibility to reveal the regulation of a small number of cellular translation levels.

The additional aspects and advantages of the present invention will be partly given in the following description, and part of them will become obvious from the following description, or be understood through the practice of the present invention.

Description of the drawings

The above and/or additional aspects and advantages of the present invention will become obvious and easy to understand from the description of the embodiments in conjunction with the following drawings, in which:

Figure 1 shows a schematic diagram of sequencing results analysis according to an embodiment of the present invention, where A is the proportion of periodic fragments in the sequencing data; B is the distribution proportion of the fragments compared to the gene after sequencing in the coding region; C It is the distribution ratio of the fragments compared to the gene after sequencing in the 5'and 3'non-coding regions;

Figure 2 shows a schematic diagram of cell number analysis required by different methods according to an embodiment of the present invention;

Figure 3 shows a schematic diagram of analyzing the number of RNAs required for library construction according to different methods according to an embodiment of the present invention;

Fig. 4 shows a schematic diagram of the influence analysis of different centrifugal conditions on the proportion of periodic fragments in the sequencing data according to an embodiment of the present invention.

Detailed ways

The solution of the present invention will be explained below in conjunction with examples. Those skilled in the art will understand that the following embodiments are only used to illustrate the present invention and should not be regarded as limiting the scope of the present invention. Where specific techniques or conditions are not indicated in the examples, the procedures shall be carried out in accordance with the techniques or conditions described in the literature in the field or in accordance with the product specification. The reagents or instruments used without the manufacturer's indication are all conventional products that are commercially available.

Example 1

1. Reagent preparation

1. Polysome buffer

20mM Tris-HCl, pH=7.4

150mM NaCl

5mM MgCl ₂

1mM DTT

100μg/ml CHX

2. Lysis solution

1×polyribosome buffer

1 mass% Triton-X 100

25U/ml Turbo DNase

3. Sucrose buffer solution

1×polyribosome buffer

1M sucrose

20U/ml SUPERase.In

4. RNA gel extraction buffer

300mM sodium acetate, pH value is 5.51M NaCl

0.05% by mass Tween

0.5mM EDTA

5. Clump buffer

1% by mass SDS

10mMTirs, pH=7.5

2. Preparation of cell lysate

1. Place the cell sample at the bottom of the 1.5ml EP tube.

2. Add 310μl of ice-cold lysis buffer to the EP tube.

3. Mix properly, invert the EP tube back and forth, and incubate the EP tube on ice for 10 minutes.

4. Centrifuge at 20,000g for 10 minutes in a centrifuge at 4°C, and carefully recover the supernatant.

5. Put the supernatant in a new EP tube.

3. RNA nuclease digestion and ribosome purification and recovery

6. Add 1μl RNase I (100U/μl) to the supernatant. At 22℃, use a constant temperature mixer (Eppendorf

), 1000rpm, gently shaking for 45 minutes.

7. Add 10μl SUPERase·in, which is RNase inhibitor, to stop nuclease digestion.

8. Prepare an ultracentrifuge tube (1ml centrifuge tube adapted to the Beckman MLA-150 rotor), first draw 0.7ml sucrose buffer solution and place it at the bottom of the tube, slowly transfer 300μL of the lysate from the previous step to the top of the sucrose solution, and use Mark Mark a line vertically with the pen on one side of the tube, and align the line with the outer edge of the centrifuge when placing the centrifuge tube in the rotor.

9. Use a desktop ultracentrifuge (Optima MAX-XP, Beckman), an MLA-150 rotor (Beckman), and a matching 1ml centrifuge tube (Beckman) to centrifuge at 4°C and 260,000g for 4 hours to precipitate ribosomes.

10. Remove the centrifuge tube from the rotor and aspirate the supernatant in the tube.

11. Rinse the ribosome clumps with 50μL clump buffer until they are completely dissolved, then transfer to a new 1.5ml EP tube.

12. Add 1ml TRIzol, mix well, and place at room temperature for 5 minutes; then add 0.2ml of chloroform, mix well and place at room temperature for 2 minutes.

13. Transfer the solution to Invitrogen ^TM Phasemaker ^TM Tubes, and centrifuge for 10 minutes at 4°C and 12,000 g in a centrifuge.

14. Transfer the upper aqueous phase solution to a new EP tube, add 0.75 ml of isopropanol and 1 μL of glycogen (RNA grade).

15. Place it at -20°C for 30 minutes, and centrifuge for 40 minutes at 4°C and the highest speed in a centrifuge. Aspirate the liquid and float the precipitate with 80% ethanol. Centrifuge for 5 minutes and aspirate the supernatant. Open the lid and let the liver glycogen pellets air dry.

16. Dissolve liver glycogen with 6μL RNase-free water to obtain RNA protected by ribosomes. At this time, RNA can be stored at -20°C for a week or at -80°C for several months.

Fourth, the purification of ribosomal protection fragments

17. Prepare 15% urea-TBE-PAGE gel, rinse the sample hole of TBE gel, and blow off excess urea. Perform electrophoresis in 1×TBE solution at 210V for 20 minutes in advance.

18. Add 6 μL of 2× Denaturing Loading Buffer (Thermon) to each sample.

19. Denature the sample for 90 seconds at 80°C.

20. Carefully rinse the sample hole again and load the sample. Only one sample is loaded per piece of glue, and no marker solution is loaded.

21. 210V, electrophoresis separation for 60 minutes, until the dark blue dye runs out of the gel.

22. Determine the lane where the sample is located. Starting from the light blue dye, cut out a rubber block about a little finger-wide and put it into a RNase-free 1.5ml EP tube. (Note: In order to prevent contamination between samples, a blade is used for each sample.)

23. (Pre-experimental determination range) Cut off the 29-nt to 34-nt region defined by RNA marder and put it into a new EP tube.

24. Extract RNA from polyacrylamide gel slices

(i) Add 400μL of RNA gel extraction buffer and place it at -80 for 30 minutes.

(ii) The sample is gently mixed overnight in a constant temperature mixer at 22°C at 1000 rpm.

(iii) Centrifuge briefly, collect the liquid at the bottom of the tube, and transfer 400 μL of the liquid to a clean EP tube.

25. Add 500μL of isopropanol, then add 1μL of Glycogen, and mix by inversion to precipitate RNA. Precipitate and purify RNA as described in steps 15 and 16.

26. Dissolve the RNA in RNase-free water. At this time, the RNA can be stored at -20°C for one month or at -80°C indefinitely.

Fifth, the method of building a database based on the CAT Small RNA-seq kit of diagenode

27. Build a library of dissolved RNA.

28. After building the library, use

Beads purify the PCR product and remove the remaining PCR primers.

29. Perform next-generation sequencing on the resulting library.

Refer to Figure 1 for the analysis of the sequencing results using the present invention. Periodicity is a very important indicator of ribosome map sequencing. If a certain percentage of fragments in the data are periodic, the data quality is good. The higher the ratio, the better the data quality. Generally, about 50% is the best. Compared with the data published earlier by Ingolia (Ingolia et al., 2011 ^[1] ), the quality of the data obtained by this experimental method is excellent (Figure 1A), and the sequencing data obtained by the Ingolia method does not contain periodic fragments. In the ribosome map data, the higher the ratio of the gene coding region (CDS), the better the data quality, generally between 50% and 80%. Compared with mRNA sequencing data, the ratio of ribosomal maps in the untranslated region will be significantly reduced, generally less than 5% is better, and the ratio of 3'UTR is often lower than that of 5'UTR. This figure shows that the quality of the data obtained by this experimental method is excellent, the proportion of each sample in the coding area is higher than 70%, the proportion of 3'UTR is lower than 5'UTR, and both are lower than 5% (Figure 1B and C ).

Example 2

The three commonly used ribosome mapping techniques include: Glincy et al., 2017 ^[2] , Hornstein et al., 2016 ^[3] and Reid et al., 2015 ^[4] . Refer to Figure 2 for the number of cells required by these three technologies and the method of Example 1. It can be seen that the number of cells required by this method has been significantly reduced, a total of several orders of magnitude. Therefore, the method of the present invention A very small number of cells can be used to sequence RNA fragments protected by ribosomes. In addition, referring to Fig. 3, the amount of RNA protected by ribosomes required for library construction of the present invention is small, which helps the application of ribosomal mapping technology in a small number of cellular processes.

Example 3

In step 9 of step 3 of Example 1, the influence of different centrifugation conditions on the quality of sequencing data was compared. Among them, the centrifugation condition of the control group was centrifugation for 1 hour under the condition of 100,0000g. As a result, as shown in FIG. 4, the sequencing data obtained by using the centrifugal conditions of the present invention contains a higher proportion of periodic fragments, indicating that the quality of sequencing data is better.

references

[1]Ingolia,N.T.,Lareau,L.F.,and Weissman,J.S.(2011).Ribosome profiling of mouse embryonic stem cells reveals the complexity and dynamics of mammalian proteomes.Cell 147,789-802.

[2]Glincy,N.J.M.,and Ingolia,N.T.(2017).Transcriptome-wide measurement of translation by ribosome profiling.Methods.

[3]Hornstein,N.,Torres,D.,Das Sharma,S.,Tang,G.,Canoll,P.,and Sims,PA(2016).Ligation-free ribosome profiling of cell type-specific translation in the brain.Genome Biol. 17,149.

[4]Reid,D.W.,Shenolikar,S.,and Nicchitta,C.V(2015).Simple and Inexpensive ribosome profiling analysis of mRNA translation.91,69–74.

In the description of this specification, descriptions with reference to the terms "one embodiment", "some embodiments", "examples", "specific examples", or "some examples" etc. mean specific features described in conjunction with the embodiment or example , Structure, materials or features are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the described specific features, structures, materials or characteristics can be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art can combine and combine the different embodiments or examples and the characteristics of the different embodiments or examples described in this specification without contradicting each other.

Although the embodiments of the present invention have been shown and described above, it can be understood that the above-mentioned embodiments are exemplary and should not be construed as limiting the present invention. Those of ordinary skill in the art can comment on the above-mentioned The embodiment undergoes changes, modifications, substitutions and modifications.

Claims

A method for sequencing RNA fragments protected by ribosomes, which is characterized in that it comprises:

(1) The cells are lysed to obtain a lysate;

(2) Using ribonuclease to digest the lysate, and add the obtained digestion solution to the surface of the sucrose buffer, perform centrifugation, and collect the precipitate;

(3) Purifying the precipitate to obtain a ribosome-protected RNA crude extract;

(4) Perform electrophoresis on the crude extract, collect gel slices in a predetermined area, and extract RNA protected by ribosomes from the gel slices; and

(5) Building and sequencing the RNA protected by the ribosome;

Wherein, in step (1), the number of the cells is 50-1×10 6 cells.
The method according to claim 1, wherein in step (2), the lysate is digested with RNase I enzyme,

Wherein, the dosage of the RNase I enzyme is 0.5-1.5 μL, preferably 1.0 μL.
The method according to claim 1, wherein step (2) further comprises:

Add the sucrose buffer to the centrifuge tube, then transfer the digestion solution to the surface of the sucrose buffer, and centrifuge at 200,000-350,000 g at 2-6°C for 2-6 hours. Aspirate the supernatant in the medium, wash the pellet with clump buffer until the pellet is dissolved;

Wherein, the agglomerate buffer includes: a Tris buffer containing 0.5 to 1.5% by mass of SDS, a concentration of 5 to 15 mM, and a pH of 7 to 8.
The method according to claim 1, wherein in step (3), the purification treatment comprises:

Mix the precipitate with TRIzol and chloroform in sequence and let it stand, and centrifuge the resulting mixture to collect the supernatant;

Add isopropanol and glycogen to the supernatant, let stand, centrifuge, discard the supernatant, suspend the precipitate with ethanol, centrifuge, discard the supernatant, dry the precipitate, and then dissolve the precipitate with water , In order to obtain the ribosome-protected RNA crude extract.
The method according to claim 1, characterized in that, in step (4), a gel is used for electrophoresis, and one kind of the crude extract is loaded on each gel, and no marker solution is loaded.
The method according to claim 1, characterized in that, in step (5), the CAT Small RNA-seq kit of diagenode company is used to construct the database.
A method for sequencing RNA fragments protected by ribosomes, which is characterized in that it comprises:

(1) Provide the following reagents:

Polyribosome buffer, including: 20mM Tris-HCl solution, pH=7.4; 150mM NaCl solution; 5mM MgCl 2 solution, 1mM dithiothreitol solution; 100μg/ml cycloheximide solution;

Lysis buffer, including: 1× the polysome buffer, 1% by mass Triton-X 100 solution; 25 U/ml deoxyribonuclease;

Sucrose buffer solution, including: 1×polyribosome buffer; 1M sucrose solution; 20U/ml RNase inhibitor;

RNA gel extraction buffer, including: 300mM sodium acetate solution; 1M NaCl solution; 0.05% by mass Tween; 0.5mM EDTA;

Agglomerate buffer, including: 1% by mass SDS solution; 10mM Tirs solution, pH=7.5;

(2) Put 50～1×10 6 cells in a centrifuge tube;

(3) Add 310 μl of ice-cold lysis buffer to the centrifuge tube, mix well, invert the centrifuge tube back and forth, and then incubate the centrifuge tube on ice;

(4) Place the centrifuge tube in a centrifuge at 4°C, centrifuge at 20,000g for 10 minutes, recover the supernatant and place it in a new centrifuge tube;

(5) Add 1 μl of RNase I to the supernatant, and shake it with a constant temperature mixer at 1000 rpm for 45 minutes at 22°C;

(6) Add 10μl of RNase inhibitor, stop nuclease digestion, and obtain lysate;

(7) First take 0.7ml sucrose buffer solution and place it at the bottom of the centrifuge tube, slowly transfer 300μL of the lysate from the previous step to the top of the sucrose solution, and place the centrifuge tube in the rotor of the centrifuge at 4℃, 260,000g. Centrifuge for 4 hours to precipitate ribosomes;

(8) Remove the centrifuge tube from the rotor, discard the supernatant in the tube, wash the place where the ribosome clumps are with 50μL clump buffer until they are completely dissolved, then transfer the lysate to a new centrifuge Tube in

(9) Add 1ml of TRIzol, mix well, leave at room temperature for 5 minutes, then add 0.2ml of chloroform, after mixing, leave at room temperature for 2 minutes, centrifuge at 4℃, 12,000g for 10 minutes, collect the supernatant in a new In the centrifuge tube, add 0.75ml of isopropanol and 1μL of liver glycogen to the centrifuge tube;

(10) Place at -20°C for 30 minutes or overnight; centrifuge for 40 minutes at 4°C and maximum speed in a centrifuge, discard the liquid, float the precipitate with 80% ethanol, centrifuge for 5 minutes and discard it The supernatant, open the lid, and dry the precipitate;

(11) Dissolve the precipitate with 6μL of RNase-free water to obtain the ribosome-protected RNA crude extract;

(12) Prepare 15% urea-TBE-PAGE gel, rinse the sample hole of TBE gel, blow off excess urea, and perform electrophoresis in 1×TBE solution at 210V for 20 minutes in advance;

(13) Add 6 μL 2× denaturation loading buffer to the crude extract, and denature the sample at 80°C for 90 seconds;

(14) Rinse the sample hole again, load the sample after the previous step of denaturation treatment, and load only one piece of glue for each sample, and no marker solution;

(15) Electrophoresis is separated under 210V voltage for 60 minutes, and electrophoresis is stopped when the dark blue dye runs out of the gel;

(16) Determine the lane where the sample is located, start from the light blue dye, cut a piece of glue, and put it into a 1.5ml centrifuge tube without RNase;

(17) Cut off the 29-34 nt RNA aggregation area on the gel block and put it into a new centrifuge tube;

(18) Add 400μL RNA gel extraction buffer to the centrifuge tube, and place it at -80 for 30 minutes;

(19) Place the centrifuge tube in a constant temperature mixer at 22°C at 1000 rpm and gently mix overnight;

(20) Centrifuge, collect the liquid at the bottom of the tube, and transfer 400 μL of liquid to a clean centrifuge tube;

(21) Add 500 μL of isopropanol to the centrifuge tube, then add 1 μL of glycogen, and mix upside down, and repeat steps (10) and (11) to purify RNA;

(22) Use diagenode's CAT Small RNA-seq kit to build a library of dissolved RNA and sequence the obtained library.